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Proximity ligation in situ assay (PLISA) is a powerful method to quantify endogen protein-protein interactions in cells and simultaneously identify localization of these interactions. PLISA can be used to quantify autophagy flux and can as well be adapted to assess global autophagy (SQSTM1/P62-LC3B interaction) or specific autophagy, such as mitophagy (NIX-LC3B). Here, we describe a step-by-step method to monitor autophagy using PLISA in adherent cancer cells.

The Atg8-family proteins are subdivided into two subfamilies: the GABARAP and LC3 subfamilies. These proteins, which are major players of the autophagy pathway, present a conserved glycine in their C-terminus necessary for their association to the autophagosome membrane. This family of proteins present multiple roles from autophagy induction to autophagosome-lysosome fusion and have been described to play a role during cancer progression. Indeed, GABARAPs are described to be downregulated in cancers, and high expression has been linked to a good prognosis. Regarding LC3 s, their expression does not correlate to a particular tumor type or stage. The involvement of Atg8-family proteins during cancer, therefore, remains unclear, and it appears that their anti-tumor role may be associated with their implication in selective protein degradation by autophagy but might also be independent, in some cases, of their conjugation to autophagosomes. In this review, we will then focus on the involvement of GABARAP and LC3 subfamilies during autophagy and cancer and highlight the similarities but also the differences of action of each subfamily member. Abbreviations: AIM: Atg8-interacting motif; AMPK: adenosine monophosphate-associated protein kinase; ATG: autophagy-related; BECN1: beclin 1; BIRC6/BRUCE: baculoviral IAP repeat containing 6; BNIP3L/NIX: BCL2 interacting protein 3 like; GABARAP: GABA type A receptor-associated protein; GABARAPL1/2: GABA type A receptor associated protein like 1/2; GABRA/GABA(A): gamma-aminobutyric acid type A receptor subunit; LAP: LC3-associated phagocytosis; LMNB1: lamin B1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; PI4K2A/PI4KIIα: phosphatidylinositol 4-kinase type 2 alpha; PLEKHM1: plecktrin homology and RUN domain containing M1; PtdIns3K-C1: class III phosphatidylinositol 3-kinase complex 1; SQSTM1: sequestosome 1; ULK1: unc51-like autophagy activating kinase 1.

EMT is a reversible cellular process that is linked to gene expression reprogramming, which allows for epithelial cells to undergo a phenotypic switch to acquire mesenchymal properties. EMT is associated with cancer progression and cancer therapeutic resistance and it is known that, during the EMT, many stress response pathways, such as autophagy and NMD, are dysregulated. Therefore, our goal was to study the regulation of ATG8 family members (GABARAP, GABARAPL1, LC3B) by the NMD and to identify molecular links between these two cellular processes that are involved in tumor development and metastasis formation. IHC experiments, which were conducted in a cohort of patients presenting lung adenocarcinomas, showed high GABARAPL1 and low UPF1 levels in EMT+ tumors. We observed increased levels of GABARAPL1 correlated with decreased levels of NMD factors in A549 cells in vitro. We then confirmed that GABARAPL1 mRNA was indeed targeted by the NMD in a 3′UTR-dependent manner and we identified four overlapping binding sites for UPF1 and eIF4A3 that are potentially involved in the recognition of this transcript by the NMD pathway. Our study suggests that 3′UTR-dependent NMD might be an important mechanism that is involved in the induction of autophagy and could represent a promising target in the development of new anti-cancer therapies.

Autophagy is a catabolic process in which cytosolic constituents such as damaged proteins and organelles are degraded by lysosomal hydrolases and this pathway therefore recycles nutrients and provides energy to the cells. In this review, we will describe the involvement of autophagy in antigen presentation and its impact on cancer immunotherapy. In antigen presenting cells, the autophagy pathway generates peptides from endogenous antigen presented by the major histocompatibility class II (MHC-II) proteins to CD4+ T cells. The contribution of CD4+ T cells in the immune response is crucial and central since they coordinate and increase the activity of every cell types involved in immunity and especially in the immunity against cancer. Autophagy is also important in antigen donor cells like for instance tumor cells. Indeed, when captured by antigen presenting cells, these exogenous antigens will be more efficiently presented on MHC-I by the mechanism of cross-presentation for a stronger priming of CD8+ T cells and a better cytotoxic activity. Finally, the involvement of autophagy in antigen presentation led to consider the modulation of autophagy as a mean to improve immunotherapy through three promising axes. Firstly, the release of autophagosomes by tumor cells treated with proteasome and autophagosome-lysosome fusion inhibitors or autophagic inducer is a source of tumor antigens for therapeutic vaccination. Secondly, fusion proteins between ATG8 family proteins or cargo adaptors and tumor antigens will specifically address the tumor antigens into autophagosomes in order to facilitate their MHC-II presentation and efficiently prime CD4+ T cells. Finally, the use of radiotherapy can increase autophagy in tumor cells, enhances MHC-I expression level at the surface of tumor cells making them better target for CD8+ T cell cytotoxicity.

Simple Summary Epithelial–mesenchymal transition (EMT) is involved in metastasis formation, chemoresistance, apoptosis resistance, and acquisition of stem cell properties, making this process an attractive target in cancer. However, direct targeting of EMT remains challenging. Autophagy—an intracellular mechanism—has been noted to be involved in the regulation of EMT—mainly by its involvement in the degradation of EMT actors, explaining why understanding of how autophagy could regulate EMT might be promising in the development of new cancer therapies. Here, we found that GABARAPL1—an autophagy-related gene—was increased in human NSCLC mesenchymal tumors compared to epithelial tumors, and induction of EMT in an A549 lung cancer cell line by TGF-β/TNF-α cytokines also led to an increase in GABARAPL1 expression. This regulation could involve the EMT-related transcription factors of the SMAD family. To understand the role of GABARAPL1 in EMT regulation in lung cancer cells, A549 KO GABARAPL1 were designed and used to investigate whether GABARAPL1 could inhibit EMT via its involvement in SMAD degradation. The results indicate that GABARAPL1-mediated autophagic degradation could intervene as a negative EMT-regulatory loop. Abstract The pathway of selective autophagy, leading to a targeted elimination of specific intracellular components, is mediated by the ATG8 proteins, and has been previously suggested to be involved in the regulation of the Epithelial–mesenchymal transition (EMT) during cancer’s etiology. However, the molecular factors and steps of selective autophagy occurring during EMT remain unclear. We therefore analyzed a cohort of lung adenocarcinoma tumors using transcriptome analysis and immunohistochemistry, and found that the expression of ATG8 genes is correlated with that of EMT-related genes, and that GABARAPL1 protein levels are increased in EMT+ tumors compared to EMT- ones. Similarly, the induction of EMT in the A549 lung adenocarcinoma cell line using TGF-β/TNF-α led to a high increase in GABARAPL1 expression mediated by the EMT-related transcription factors of the SMAD family, whereas the other ATG8 genes were less modified. To determine the role of GABARAPL1 during EMT, we used the CRISPR/Cas9 technology in A549 and ACHN kidney adenocarcinoma cell lines to deplete GABARAPL1. We then observed that GABARAPL1 knockout induced EMT linked to a defect of GABARAPL1-mediated degradation of the SMAD proteins. These findings suggest that, during EMT, GABARAPL1 might intervene in an EMT-regulatory loop. Indeed, induction of EMT led to an increase in GABARAPL1 levels through the activation of the SMAD signaling pathway, and then GABARAPL1 induced the autophagy-selective degradation of SMAD proteins, leading to EMT inhibition.